EP0554314A1 - Verfahren zur bestimmung von porphyromonas gingivalis. - Google Patents
Verfahren zur bestimmung von porphyromonas gingivalis.Info
- Publication number
- EP0554314A1 EP0554314A1 EP91918757A EP91918757A EP0554314A1 EP 0554314 A1 EP0554314 A1 EP 0554314A1 EP 91918757 A EP91918757 A EP 91918757A EP 91918757 A EP91918757 A EP 91918757A EP 0554314 A1 EP0554314 A1 EP 0554314A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- gingivalis
- glycine
- activity
- sample
- tripeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/10—Anilides
- C12Q2337/12—Para-Nitroanilides p-NA
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
Definitions
- the present invention is related to a method of determining proteolytic activity of an enzyme from Porphyro onas gin- givalis, formerly named Bacteroides gingivalis, below referred to as P. gingivalis.
- An object of the invention is to facilitate and/or reduce the cost of qualitative and quantitative determination of P. gingivalis in diagnosis of periodontitis and/or in laboratory techniques where proteolytic enzymes from P. gingivalis occur, especially where such enzymes occur in very small amounts.
- Periodontal diseases are caused by bacteria.
- Recent clinical and microbial investigations have revealed that specific gram negative bacteria play an ethological role in different forms of human periodontal diseases.
- Porphyromonas gingivalis has been associated with progressive adult periodontitis.
- the presence of P. gingivalis could be used as a guideline for selection of treatment regime and for treatment evaluation.
- Identi ⁇ fication of P. gingivalis is commonly performed using cultivation techniques. Microbial sampling and culturing is usually performed as in the following example:
- Sterile paperpoints are inserted into the periodontal pocket and left in place for 15 s.
- the points are p o oled in a vial containing glass beads and transport medium.
- the samples are thoroughly desintegrated and diluted.
- a volume of 0.1 ml from each dilution as well as from the undiluted sample is distributed on the surface of Brucella agar plates (BBL Microbiology Systems, Cockeysville, MD, USA) enriched with 5% defibrinated horse blood, 0.5% haemolyzed blood and 5 mg/1 of menadion. After 7-9 days of incubation, in jars with 95% H and 5% CO., total viable count and the number of P.
- gingivalis colonies in the sample is determined.
- the P. gingivalis colonies are identified by gram staining, lactose fermentation test, and checking for production of red fluorescence in long-wave (360 nm) UV-light.
- the handling of the sample is time consuming and personnel-intensive and therefore expen ⁇ sive. Further, it will take about 2 weeks before the therapist can have the result of the test.
- P. gingivalis is known to produce high proteolytic activity, which can a) destroy in vitro many components of connective tissue and b) degrade other proteins important for the maintenance of local homeostasis e.g. the inactivation of inhibitors controlling host proteinases.
- EP-A2 0 255 341 discloses a periodontal diagnosis reagent, which comprises peptide(s) having attached thereto a colour developing group for detecting aminopeptidase-like enzymatic activity in an oral sample.
- EP-A2 0 292 708 (The Research Foundation of State University of New York) relates to detection of Bacteroides gingivalis using a serum aminopeptidase inhibitor in an assay system, and measuring the ability to hydrolyse N-carbobenzoxy- glycyl-glycyl-L-arginine- ⁇ -naphtylamide derivatives.
- enhancers of the enzymatic activity of Bacteroides gingivalis such as chelating materials, specifically tetrasodium ethylenediaminetetraacetate (EDTA) is proposed.
- EDTA tetrasodium ethylenediaminetetraacetate
- the present invention relates to a method of determining P. gingivalis in diagnosis and/or treatment of periodon ⁇ titis, whereby a sample is taken from a possible site of P. gingivalis and said sample is added to a composition comprising a substrate subject to proteolysis, amidolysis or proteolysis, and at least one activator for the proteolytic, amidolytic or esterolytic activity of one or more enzymes from P.
- said activator being cysteine, cysteamine, glycine, or a di- or tripeptide containing glycine, or an amide or a lower alkyl ester of glycine or such di- or tripeptide, or a decarboxylated derivative of such di- or tripeptide; and determining proteolytic, amidolytic or esterolytic activity from said sample in said composition.
- the invention relats to a kit for determining of P. gingivalis in diagnosis and/or treatment of periodontitis, comprising a sampling device for collection of a sample from a possible site of P. gingivalis, said kit further comprising a composition comprising a substrate subject to proteolysis, amidolysis or esterolysis, and at least one activator for the proteolytic, amidolytic or esterolytic activity of one or more enzymes from P.
- said activator being cysteine, cysteamine, glycine, or a di- or tripeptide containing glycine, or an amide or a lower alkyl ester of glycine or such di- or tripeptide, or a decarboxylated derivative of such di- or tripeptide.
- Preferred substrates for use according to the invention are D- or L-amino acids or peptides of 2-6 D- or L-amino acids having arginine at the PI position, to which a chromogenic or fluorogenic group is attached by an amide or an ester bond, e.g.
- the activator is other than glycine, since the proteolysis-activating effect of glycine is comparatively low.
- cysteamine is non-preferred as sole activator, since it requires a concentration of at least 200 mM for sufficient effect.
- reducing agents such as cysteamine and mercaptoethanol are preferably used along with one of the activators (other than cysteamine) .
- glycine In di- and tripeptides, their amides and esters, as activators, glycine may be in either position and may occur once or twice, and three times (in glycyl-glycyl- glycine, its amides and esters) .
- a sole glycine, or an acidic amino acid such as aspartic or glutamic acid, is however not the C-terminal amino acid in the di- and tri-peptides with unblocked carboxyl group.
- the amino acids in addition to glycine are selected from naturally occurring ⁇ -amino acids and their D-analogues.
- the amide of glycine or the di-or tripeptide is
- Am is the residue of glycine, di-or tri-peptide and R 1 and R2 are each selected from H or an ali.phati•c, heteroaliphatic, aromatic or heteroaromatic group containing 1-9 carbon atoms.
- R 1 and R2 are each selected from H, or an aliphatic, aromatic or heteroaromatic group containing 1-4, most preferably 1-3, carbon atoms.
- R and R 2 are each selected from H or an phenyl, 2-, 3-, 4-pyr ⁇ dyl or a 9-antracene group.
- R 2 i.s H, and most preferably R1 and R2 are both H.
- the ester is Am - OR 3 wherein Am is the residue of glycine, di- or tri-peptide and R 3 i.s an alkyl or alkenyl group containing 1-5 carbon atoms.
- the best embodiment of the invention known to the inventor is employing Gly-NH_ and cysteamine in a buffer, in a device as shown below in Fig. 4A.
- concentration of activator in the composition employed by the invention is suitably 10 to 200 mM preferably 20 to 100 mM.
- Fig. 1 is a diagram showing pH dependent activity of P. gingivalis crude extracts.
- Fig. 2A is a diagram showing relative stimulatory (activat ⁇ ing) effect of various amino acids and an amide, while
- Fig. 2B is a diagram showing relative stimulatory (activat ⁇ ing) effect of various peptides and derivatives thereof.
- Fig. 3 contains three diagrams (A) , (B) and (C) showing the effect of glycine-containing compounds and/or reducing agents on the amidolytic activity of P. gingivalis pro- teinases.
- Fig. 4A is a schematic view from the above side of a diagnostic test kit according to one specific embodiment of the invention.
- Fig. 4B is a schematic view of the kit in Fig. 4A shown from the below side thereof, and
- Fig. 4C is a schematic view from above of a diagnostic test kit according to another specific embodiment of the invention.
- Fig. 1 Illustrated in Fig. 1 is the effect of glycyl-glycine on pH-dependent hydrolysis of BAPNA by proteinases present in P. gingivalis crude extracts. Samples were incubated in either 0.2 M Bis-Tris-HCl buffer (pH 6.0 - 7.2) or 0.2 M Tris-HCl buffer (pH 7.4 - 9.0), both containing 5 mM CaCl , 5 mM cysteine, in the absence (solid circles) or presence (open circles) of 50 mM glycyl-glycine, and assayed for amidase activity using 1 mM BAPNA.
- Fig. 1 Illustrated in Fig. 1 is the effect of glycyl-glycine on pH-dependent hydrolysis of BAPNA by proteinases present in P. gingivalis crude extracts. Samples were incubated in either 0.2 M Bis-Tris-HCl buffer (pH 6.0 - 7.2) or
- 2A and 2B illustrate the effect of amino acids and dipeptides, respectively, in stimulating the amidase activity of crude extracts and purified proteinases from P. gingivalis and clostripain from Clostridium histolyticum.
- Samples were incubated in a buffer system composed of 0.2M Tris-HCl, 5 mM CaCl,, 5 mM cysteine, pH 7.4 in the presence of 50 mM concentrations of each compound tested and then tested against BAPNA (1 mM) . Controls contained only buffer and the activity generated given an activity value of 1.0.
- Aspartane Aspartane (Asp-Phe-OMe) was not tested against the 17 kD proteinase.
- (A) effect of amino acids and glycylamide
- (B) effect of glycine-containing dipep ⁇ tides and aspartane-related compounds.
- Fig. 3A, 3B and 3C Effect of glycine-containing compounds and/or reducing agents on the amidolytic activity of P. gingivalis proteinases.
- Samples were incubated in 0.2M Tris-HCl, 5 mM CaCl , pH 7.5, in the presence of either low (Fig. 3A) or high (Fig. 3B) concentrations of reducing agents.
- Assays for residual amidolytic activity using BAPNA (1 mM) were then measured in the absence (Figs. 3A and 3B) or presence (Fig. 3C) of glycine-containing compounds.
- Fig. 3A, 3B and 3C Effect of glycine-containing compounds and/or reducing agents on the amidolytic activity of P. gingivalis proteinases.
- Samples were incubated in 0.2M Tris-HCl, 5 mM CaCl , pH 7.5, in the presence of either low (Fig. 3A) or high (Fig. 3B) concentrations of
- ⁇ represents cysteine
- ⁇ represents 2-mercaptoethanol
- O represents dithiothreitol
- Q represents cysteamine
- Illustrated in Fig. 3C are incubations made in the presence of various reducing agents plus either glycinamide (1200 mM) (open symbols) or glycyl-glycine (closed symbols) , whereby ( ⁇ £>, ⁇ ) represent cysteine; (A/A) represent 2- mercaptoethanol and (O f l) represent dithiothreitol.
- 1 is a blister card made of rigid blister material having securely and leak-free but releasab- ly attached thereto a plastic-coated aluminium foil cover 2, having a released end portion 2A.
- Hinted at 3 is a portion of 2 covering a sampling stick blister 5
- hinted at 4 is a portion of 2 covering a blister 6 for holding a liquid or jelly composition.
- Items 5 and 6 are vacuum formed from 1.
- Item 7 is a sampling stick having a point 8 on which a sample may be adsorbed.
- Denoted 9 is a liquid or jelly buffer solution containing a substrate for P. gingivalis enzyme and an activator, said substrate having a chromogenic leaving group.
- Figs. 4A and B Use of the device in Figs. 4A and B is generally as follows, Placing blister card 1 horizontally as in Fig. 4A, foil cover 2 is removed. The sampling stick 7 is taken by the broad end, and a sample is taken at point 8. Point 8 is immersed in the buffer solution 9. A colour change in buffer solution 9 indicates presence at the sampling site of P. gingivalis.
- the device of Figs. 4A and 4B may be varied by incorporating a second blister holding a liquid or jelly buffer solution containing the same substrate without activator, and a second blister holding a second sampling stick.
- Said second solution and sampling stick serve for control of amidolytic activity unrelated to P. gingivalis.
- the device in Fig. 4C is similar to that in Figs. 4 A and B, and items 101 through 108, when shown, generally corre ⁇ spond to items 1 through 8 , except that buffer solution 109 does not contain a substrate with a chromogenic group. Instead, said substrate is placed on a distal portion 112 of a test strip 111 in a test strip blister 110. After sampling with 107, 108 is immersed in 109 to allow the sample to dissolve or suspend in liquid 109. Test strip 111 is taken from the end opposite to 112, and 112 is immersed in 109. A colour change on 112 indicates presence at the sampling site of P. gingivalis. EXPERIMENTAL
- the bacterial proteinase clostripain (Sigma) obtained from the anaerobe Clostridium histolyticum was totally unaffected by any of the amino acids or dipeptides tested, despite the fact that it also requires both Ca and a reducing agent for maximum activity.
- cysteine was of some significance since other reducing agents such as 2-mercaptoethanol and dithiothreitol were not nearly as effective in stimulating amidase activity in the absence of glycine containing compounds (Fig.s 3A and 3B) .
- glycyl- glycine and glycylamide maximum enhancement of amidolytic activity could readily be attained at low reducing agent concentrations (0.5mM) (Fig. 3C) .
- marked differen ⁇ ces in the degree of activation in the presence of in ⁇ dividual thiol-containing compounds was also noted with cysteine, giving a much greater stimulatory effect than that of either 2-mercaptoethanol or dithiothreitol.
- SUBSTITUTESH ⁇ HT suggested that cysteine itself, might be acting both as a reducing agent and a stimulating agent.
- thiol-containing compounds were utilized in an assay system containing no other sources of either reducing or stimulating agents.
- the effect of cysteamine was studied since the carboxyl group of glycine appeared to reduce stimulation (glycine versus glycinamide) .
- Fig. 3B cysteamine could best fulfil the dual role required for P. gingivalis amidase and proteinase activity.
- Gly-Gly or Gly-amide does not have any effect on activity of:
- Serine proteinases trypsin, chymotrypsin and elastase and subtylisin
- Metalloproteinases thermolysin, S.aureus metal- loproteinase
- Cysteine proteinases (clostripain, papain and cathepsin B) 4. Effect of Gly-Gly (50mM) and glycylamide (100 mM) on amidolytic activity of crude extract of human liver (rich source of cathepsins, amino- and carboxy-pep- (rich source of cathepsins, amino- and carboxy-pep- tidases) , pancreas (abundant in peptidases and pro ⁇ teinases) , leucocytes (HLE, cathepsin G and other cysteine cathepsins, metalloproteinases) and rabbit testicles (acrosin and cathepsins) .
- Gly-Gly 50mM
- glycylamide 100 mM
- Gly-Gly and Gly-amide are group specific and limited only to bacterial enzymes.
- Glycylamide at low concentrations and in the absence of heparin (present in tissues as a physiological tryptase activator) activates tryptase but has no effect on enzyme activity at concentration higher than 100 mM. Moreover, in the presence of heparin glycylamide acts as tryptase inhibitor.
- Gly-amide at concentration of 100 mM as a P. gingivalis proteinase stimulator is very useful because it eliminates tryptase activity (tryptase was found in crevicular fluid) which might interfere with the specificity of P. gingivalis detection in clinical samples.
- A 0.2 M Tris-HCl, 5 mM CaCl 2 , pH 7.5
- B 0.2 M Tris-HCl, 5 mM CaCl 2 , 5 mM Cys, Ph 7.5
- C 0.2 M Tris-HCl, 5 mM CaCl 2 , 5 mM Cys, 50 mM Gly-Gly, pH 7.5D.
- Fusobacterium shows some trypsin-like amidolytic activity but this activity is much lower than the one expressed by P. gingivalis (see ratio 1/10) .
- DFP Di-fluorophosphate
- DIC 3,4-dichloroisocumarin
- BAPNA hydrolysing activity in crevicular fluid is not due to host serine proteinases (should be inhibited by either DFP or DIC) or cysteine proteinases as cathepsine B, H and L which are very strongly inhibited by cystatin C.
- lysosomal cysteine proteinases are very unstable at pH above 7.
- metalloproteinases can be excluded because they do not degrade p-nitroanilide substrates. Laboratory approach to measure trypsin-like amidolytic activity of P. gingivalis proteinases.
- Buffers A) 0.2 M Tris-HCl, 5 mM CaCl 2 , pH 7.5
- the assay was performed in BSA (bovine serum albumin) - coated flat bottom wells of microtitration plates. 140 ⁇ l of buffer A or C was placed into the well and 10 ⁇ l of gingival crevicular fluid sample (150 ⁇ l of buffer in which 2-3 paper points were placed and this subsequently frozen) was added. Then, 50 ⁇ l of BAPNA (0.4 mM) diluted in the buffer to a final concentration of 0.1 mM was added.
- BSA bovine serum albumin
- the samples from the same periodontitis site are taken on two paper points.
- One paper point is placed into a first well containing 200 ⁇ l 0.2 M Tris-HCl, 5 mM CaCl_, 1.0 mM cysteine, 0.1 mM BAPNA, pH 7.5, and the second one into a second well containing 200 ⁇ l of above buffer supplemented with 100 mM Gly-amide.
- Development of yellow colour in both wells can be monitored by eye or A._ can be measured over a time of incubation at room temperature.
- Strip A will have the pad impregnated with a suitable histochemical substrate and diazonium salt.
- Bacteria species present Activity units in periodontitis site
- One unit of activity is equal to the amount of enzyme which hydrolyses 1 nmol of BAPNA during 1 h of incubation.
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Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US59781790A | 1990-10-15 | 1990-10-15 | |
US597817 | 1990-10-15 | ||
PCT/SE1991/000695 WO1992007086A1 (en) | 1990-10-15 | 1991-10-15 | A method of determining porphyromonas gingivalis |
Publications (2)
Publication Number | Publication Date |
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EP0554314A1 true EP0554314A1 (de) | 1993-08-11 |
EP0554314B1 EP0554314B1 (de) | 1996-08-14 |
Family
ID=24393048
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP91918757A Expired - Lifetime EP0554314B1 (de) | 1990-10-15 | 1991-10-15 | Verfahren zur bestimmung von porphyromonas gingivalis |
Country Status (8)
Country | Link |
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US (1) | US5981164A (de) |
EP (1) | EP0554314B1 (de) |
AT (1) | ATE141334T1 (de) |
AU (1) | AU8765491A (de) |
DE (1) | DE69121412T2 (de) |
DK (1) | DK0554314T3 (de) |
ES (1) | ES2091335T3 (de) |
WO (1) | WO1992007086A1 (de) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2267753A (en) * | 1992-06-12 | 1993-12-15 | British Tech Group | Test for periodontitis and susceptibility thereto |
EP0728017A1 (de) * | 1993-11-10 | 1996-08-28 | Bristol-Myers Squibb Company | Behandlung von bakterieninduzierten entzündlichen erkrankungen |
US6875851B1 (en) * | 1999-03-05 | 2005-04-05 | University Of Georgia Research Foundation, Inc. | Prolyl tripeptidyl peptidases nucleic acid of Porphyromonas gingivalis |
WO2002038742A2 (en) | 2000-11-08 | 2002-05-16 | The University Of Georgia Research Foundation, Inc. | Dipeptidylpeptidases and methods of use |
SE0400191D0 (sv) | 2004-01-30 | 2004-01-30 | Tendera Ab | A test kit for detecting periodontal disease |
EP3940081A4 (de) * | 2019-04-25 | 2023-01-11 | Adtec Co., Ltd. | Verfahren zur detektion von periodontopathischen bakterien |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5223403A (en) * | 1985-05-31 | 1993-06-29 | University Of Michigan | Device for diagnosing periodontal disease |
DE3783966T2 (de) * | 1986-07-29 | 1993-05-27 | Sunstar Inc | Reagens zur pruefung von periodentalen erkrankungen. |
US4994376A (en) * | 1987-05-27 | 1991-02-19 | The Research Foundation Of State University Of Ny | Detection of bacteroides gingivalis |
DE68906167T2 (de) * | 1988-01-20 | 1993-08-05 | Kyowa Medex Co Ltd | Zusammensetzung zur erkennung von paradontalen krankheiten. |
-
1991
- 1991-10-15 AT AT91918757T patent/ATE141334T1/de not_active IP Right Cessation
- 1991-10-15 ES ES91918757T patent/ES2091335T3/es not_active Expired - Lifetime
- 1991-10-15 EP EP91918757A patent/EP0554314B1/de not_active Expired - Lifetime
- 1991-10-15 DK DK91918757.5T patent/DK0554314T3/da active
- 1991-10-15 WO PCT/SE1991/000695 patent/WO1992007086A1/en active IP Right Grant
- 1991-10-15 AU AU87654/91A patent/AU8765491A/en not_active Abandoned
- 1991-10-15 DE DE69121412T patent/DE69121412T2/de not_active Expired - Fee Related
-
1995
- 1995-01-11 US US08/371,479 patent/US5981164A/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
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See references of WO9207086A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1992007086A1 (en) | 1992-04-30 |
US5981164A (en) | 1999-11-09 |
AU8765491A (en) | 1992-05-20 |
DK0554314T3 (da) | 1996-12-23 |
ATE141334T1 (de) | 1996-08-15 |
DE69121412D1 (de) | 1996-09-19 |
EP0554314B1 (de) | 1996-08-14 |
ES2091335T3 (es) | 1996-11-01 |
DE69121412T2 (de) | 1997-02-13 |
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